Divyashree B C, Roy Sudhir C
Molecular Biology Laboratory, ICAR-National Institute of Animal Nutrition and Physiology, Hosur Road, Adugodi, Bangalore 560030, India; Department of Biochemistry, Centre for Post Graduate Studies, Jain University, Jayanagar 3rd Block, Bangalore 560011, India.
Molecular Biology Laboratory, ICAR-National Institute of Animal Nutrition and Physiology, Hosur Road, Adugodi, Bangalore 560030, India.
Theriogenology. 2018 Jan 15;106:279-286. doi: 10.1016/j.theriogenology.2017.10.014. Epub 2017 Oct 16.
Binder of sperm-5 (BSP-5) is one of the fertility-associated proteins of cattle seminal plasma. Binding of sperm to the oviductal epithelium is mediated by BSP group of proteins. However, it is not clear, whether this protein is also involved in sperm motility. In the present study, attempts were made to characterize BSP-5 protein in both normozoospermic (NS) and asthenozoospermic (AS) Murrah buffalo (n = 18; Bubalus bubalis), Holstein Friesian (n = 8, Bos taurus) and Jersey cattle (n = 8; Bos taurus) bull seminal plasma and also study its expression pattern in these species. 1-D Western blot demonstrated three major BSP-5 immunoreactive protein bands (24.2 kDa, 20.5 kDa, and 12.3 kDa) in buffalo seminal plasma. Of these, the intensities of 24.2 and 20.5 kDa protein bands reduced significantly (P ≤ 0.05) in seminal plasma of AS group compared to that of NS group. On the contrary, the expression of 12.3 kDa protein band did not vary significantly between the groups. In Holstein Friesian seminal plasma, at least six BSP-5 immunoreactive protein bands (25.1, 23.6, 19.5, 13.8, 13.1 and 12.3 kDa) could be detected. Of these, the intensities of 23.6, 13.8/13.1 and 12.3 kDa protein bands decreased (P = 0.058, 0.111, 0.053) in AS group bulls compared to NS bulls. Holstein Friesian bull seminal plasma demonstrated a BSP-5 immunoreactive duplex protein band of 13.8/13.1 kDa, which was not evident in buffalo seminal plasma. In 2-D Western blot, a train of five BSP-5 immunoreactive duplex protein spots (Mr 21.0-27.6 kDa, pI of ∼3.9-5.1) was detected. Mass spectrometry of one of the representative duplex spot confirmed that these were BSP-5 and BSP-3 proteins, respectively. Indirect immunofluorescence studies showed that BSP-5 is primarily localized to the mid-piece/mitochondrial region of buffalo spermatozoa. To conclude, the findings of the present study could establish the significance and association of BSP-5 proteins in sperm motility and how their level differ in semen from two different clinical groups of buffalo bull (NS vs. AS). Further, the study also demonstrated that the expression pattern of BSP-5 and other BSP variants in seminal plasma of bulls is species-specific.
精子结合蛋白5（BSP-5）是牛精液浆中与生育相关的蛋白质之一。精子与输卵管上皮的结合由BSP蛋白组介导。然而，尚不清楚该蛋白是否也参与精子运动。在本研究中，试图对正常精子症（NS）和弱精子症（AS）的穆拉水牛（n = 18；水牛属）、荷斯坦弗里生牛（n = 8，牛属）和泽西牛（n = 8；牛属）公牛精液浆中的BSP-5蛋白进行表征，并研究其在这些物种中的表达模式。一维蛋白质免疫印迹法显示，水牛精液浆中有三条主要的BSP-5免疫反应蛋白条带（24.2 kDa、20.5 kDa和12.3 kDa）。其中，与NS组相比，AS组精液浆中24.2 kDa和20.5 kDa蛋白条带的强度显著降低（P≤0.05）。相反，12.3 kDa蛋白条带在两组之间的表达没有显著差异。在荷斯坦弗里生牛精液浆中，可检测到至少六条BSP-5免疫反应蛋白条带（25.1、23.6、19.5、13.8、13.1和12.3 kDa）。其中，与NS公牛相比，AS组公牛精液浆中23.6、13.8/13.1和12.3 kDa蛋白条带的强度降低（P = 0.058、0.111、0.053）。荷斯坦弗里生牛精液浆显示出一条13.8/13.1 kDa的BSP-5免疫反应双链蛋白条带，这在水牛精液浆中不明显。在二维蛋白质免疫印迹法中，检测到一系列五个BSP-5免疫反应双链蛋白斑点（分子量21.0 - 27.6 kDa，等电点约为3.9 - 5.1）。对其中一个代表性双链斑点进行质谱分析证实，这些分别是BSP-5和BSP-3蛋白。间接免疫荧光研究表明，BSP-5主要定位于水牛精子的中段/线粒体区域。总之，本研究结果可以确定BSP-5蛋白在精子运动中的意义和关联，以及它们在水牛公牛两个不同临床组（NS与AS）精液中的水平差异。此外，该研究还表明，公牛精液浆中BSP-5和其他BSP变体的表达模式具有物种特异性。
